Cyclosporin A (Cyclosporine) is a potent immunosuppressant that has been widely used in the United States and other countries to prevent the rejection of transplanted organs such as kidney, heart, bone marrow, and liver in humans. The effectiveness of cyclosporin A in the treatment of other conditions (autoimmune diseases, diabetes, malaria) is being investigated.
To prevent allograft rejections, a minimum level of cyclosporin A in the blood is required throughout the lifetime of the patient. Chronic high doses can result in kidney and liver damage. Distribution and metabolism of the drug varies greatly between individuals, as well as in a single individual during the course of therapy. Accordingly, monitoring cyclosporin A levels in the blood or serum of allograft recipients is considered essential to good patient management.
Cyclosporin A has the structure: ##STR1## wherein the abbreviations represent: -MeVal- a residue of N-methyl-L-valine
-MeLeu- a residue of N-methyl-L-leucine PA1 -D-Ala- a residue of D-alanine PA1 -Ala- a residue of L-alanine PA1 -Val- a residue of L-valine PA1 -Abu - a residue of L-.alpha.-aminobutyric acid PA1 -Sar- a residue of sarcosine, also known as N-methylglycine PA1 (2) where R.sup.1 is H and R.sup.2 is W.sub.r --(C.dbd.Y).sub.m Z where W and m are as defined above and where r=1, 0 and where Z is a poly(amino acid) or poly(amino acid) derivative or a fluorescent moiety, and R.sup.3 +R.sup.4 is oxo or R.sup.3 .dbd.R.sup.4 .dbd.H; or PA1 (3) where R.sup.1 .dbd.R.sup.2 .dbd.H, R.sup.3 is OH or H, and R.sup.4 is C(R.sup.5).dbd.W--(C.dbd.Y).sub.m --Z or C(R.sup.5 R.sup.6)--W.sub.r --(C.dbd.Y).sub.m Z where W, Y, m, r & Z are defined as above, and R.sup.5 and R.sup.6 are H, lower alkyl, or substituted lower alkyl. Where Z is a poly(amino acid) or poly(amino acid) derivative, the compound is an immunogen; where Z is a fluorescent moiety the compound is a tracer. In the case of (1)-(3) above the compound is a precursor for a tracer or immunogen where Z is OH, NH.sub.2, NHNH.sub.2, or OR.sub.a, SR.sub.a, NHR.sub.a, NR.sub.a R.sub.b (where R.sub.a and R.sub.b are stable chains of from 1-10 carbon atoms), SH or a leaving group such as Cl, I Br,N.sub.3, N-hydroxysuccinimidyl, 1-hydroxybenzotriazinyl or H when m.noteq.0. PA1 m is 0, 1 or 2; PA1 p is 1; PA1 R is H, lower alkyl or CH(OH)CH.sub.3 ; PA1 R.sup.1 is H or a suitable hydroxyl protecting group such as CH.sub.3 CO; PA1 W is 1-20 atoms (not including hydrogen) selected from C, N, O and S, with no more than two heteroatoms bonded together and with oxygen never bound to another oxygen or sulfur; PA1 Y is O, S, or NH; and PA1 Z is a poly(amino acid) or poly(amino acid) derivative or a fluorescent moiety. Where Z is a poly(amino acid) or poly(amino acid) derivative, the compound is an immunogen; where Z is a fluorescent moiety the compound is a tracer. Further, where Z is OH, NH.sub.2, NHNH.sub.2, OR.sub.a, SR.sub.a, NHR.sub.a, NR.sub.a R.sub.b (where R.sub.a and R.sub.b are stable chains of from 1-10 carbon atoms), SH or a leaving group such as Cl, I, Br, N.sub.3, N-hydroxysuccinimidyl, 1-hydroxybenzotriazinyl or H when m.noteq.0, the above structure represents a precursor for tracers and/or immunogens. Also, in the case of precursor compounds X may equal CH.sub.2 OH when p is 0. PA1 Y is O, S, or NH; PA1 m is 0, 1 or 2; and PA1 Z is a poly(amino acid) or poly(amino acid) derivative or a fluorescent moiety. Where Z is a poly(amino acid) or poly(amino acid) derivative, the compound is an immunogen; where Z is a fluorescent moiety the compound is a tracer. Further, where Z is OH, NH.sub.2, NHNH.sub.2, OR.sub.a, SR.sub.a, NHR.sub.a, NR.sub.a R.sub.b (where R.sub.a and R.sub.b are stable chains of from 1-10 carbon atoms), SH or a leaving group such as Cl, I, Br, N.sub.3, N-hydroxysuccinimidyl, 1-hydroxybenzotriazinyl or H when m.noteq.0, the above structure represents a precursor for tracers and/or immunogens. PA1 k is 0 or 1, with the provision that only when n is 1 may k be 0; PA1 X.dbd.CH.sub.2, CHOH or C.dbd.O when n is 0; PA1 m is 0, 1, or 2; PA1 p is 0 or 1; PA1 R is H or lower alkyl, or CH(OH)CH.sub.3 PA1 R.sup.1 is H or a suitable protecting group such as CH.sub.3 CO; PA1 Y is O, S, or NH PA1 m is 0, 1 or 2 PA1 Z is poly(amino acid)
wherein the term "residue" refers to the condensed form of the amino acid found in peptides, as is common in the art. Also, as is common in the art, the configuration of the .alpha.-amino acid is assumed to be L unless a D-configuration is specified. Cyclosporin A is a cyclic polypeptide with 11 amino acids, including an unusual 9 carbon amino acid. Cyclosporin A is described in U.S. Pat. Nos. 4,117,118 and 4,396,542.
All the metabolites of Cyclosporin A that have been identified in which the ring is still intact result from hydroxylations and dimethylations of the parent compound. [G. Maurer, H. R. Loosli, E. Schreier, B. Keller, Drug Metabolism and Disposition, 12 (1), 120-126 (1984)]. It has not yet been determined whether one or more of these metabolites is also an immunosuppressive, or the major cause of renal or hepatic toxicity. Therefore, measurement of levels of the metabolites may also be useful in patient management. Some major metabolites of cyclosporin A include metabolite 17, metabolite 18 and metabolite 21.
______________________________________ ##STR2## Metabolite R.sup.1 R.sup.2 R.sup.3 R.sup.4 R.sup.5 other ______________________________________ 17 OH CH.sub.3 H H H 18 OH CH.sub.3 H H H ##STR3## 21 H H H H H ______________________________________
Presently, two analytical methods are used routinely for the monitoring of cyclosporin A: radioimmunoassay with either a .sup.3 H or a .sup.125 I tracer; or high performance liquid chromatography. G. J. Burckart, D. M. Canafox, G. C. Yee, Drug Intelligence and Clinical Pharmacy, 20, 649-652 (1986). See P. Donatsch et al., Journal of Immunoassay 2 (1), 19-34 (1981);, W. C. Mahoney, J. W. O. F., Clinical Chemistry 31, 459-462 (1985) respectively. The RIA detects cyclosporin A and some of its metabolites, and requires the handling of radioactive substances. The HPLC method is specific for cyclosporin A but is labor intensive. Both methods require excessive time. In the case of RIA a minimum of 21/2 hours is required. In the case of HPLC a minimum of 30 minutes is necessary. This does not include extensive sample preparation time.
Fluorescence polarization is an alternative to radioactive methods of measuring the results of a competitive binding immunoassay. Fluorescence polarization techniques are based on the principle that a fluorescent compound, when excited by plane polarized light, will emit fluorescence having a degree of polarization inversely proportional to its rate of rotation. Small, unbound fluorescent molecules will rotate quickly and have a small degree of polarization. If the fluorescent compound is bound by a large molecule such as an antibody, the rate of rotation is slow and the degree of polarization is high. In a fluorescence polarization immunoassay, the compound(s) to be detected in the sample ("analyte"(s) or "ligand"(s)) competes with a similar compound that is attached to a fluorescent moiety ("ligand analog" or "tracer") for a limited number of receptor binding sites OD antibodies specific for the analyte(s) and tracer. If there is no analyte present, the tracer will be bound by the antibody and the degree of polarization will be large. For a given amount of analyte present, the amount of tracer bound to antibody will be correspondingly less, and the degree of polarization will be correspondingly lower. Thus, the amount of analyte present can be determined by measuring the degree of fluorescence polarization observed.
Fluorescence polarization techniques for immunoassays have been disclosed, (U.S. Pat. No. 4,420,568 to Wang et al.; U.S. Pat. No. 4,476,229 to Fino et al.; U.S. Pat. No. 4,510,251 to Kirkemo et al., each commonly assigned herewith, and others).
In the case of cyclosporin A, it is of importance to detect and quantify not only cyclosporin A but also the major metabolites thereof. The present invention is an advance in the art in that novel cyclosporin A derivative compounds specifically useful in forming immunogens to raise antibodies, and a fluorescence polarization assay using the antibodies is provided for the determination of cyclosporin A and its metabolites. The antibodies raised in response to the immunogens synthesized in accordance with the present invention are capable of specifically recognizing cyclosporin A together with some of its metabolites.